Use of fibrinogen multimers

ABSTRACT

Use of fibrinogen multimers having at least 6 fibrinogen units as an ingredient for a fibrin sealant.

The invention is concerned with the use of fibrinogen multimers as aningredient of a fibrin sealant and a method of producing a fibrinsealant comprising multimers.

Fibrin sealant is a generic name covering a complex of plasmaderivatives which are usually composed of proteins, which mimic thoseactive in the last stage of the coagulation cascade, from the activationof soluble fibrinogen by thrombin to the formation of insoluble proteinfibrin (Matras 1985, Sierra 1993, Spotnitz et al 1987 and Spotnitz1995). Most of the commercial fibrin sealants are composed of two majorcomponents—thrombin supplemented with Ca⁺⁺ ions and fibrinogensupplemented with varying amounts of factor XIII. Once both componentsare mixed, the thrombin splits off fibrinopeptides A and B from therespective Aa and Bb chains of fibrinogen to form fibrin monomers thatare polymerized by hydrogen bonding to form a soluble fibrin clot.Factor XIII, activated by thrombin in the presence of Ca⁺⁺ ions,catalyzes crosslinking between the fibrin molecules, increasing themechanical strength of the clot and reducing its susceptibility toproteolytic cleavage (Gladner and Nossal 1983).

Most fibrin sealants are prepared from either plasma cryoprecipitate orfrom Cohn fraction I, as sources of fibrinogen. Cryoprecipitate isobtained by thawing frozen plasma at 2-4° C., resulting in a paste thatis recovered by centrifugation. Cryoprecipitate contains fibrinogen,fibronectin, vonWillebrand factor and other proteins that areco-precipitated with the above proteins, such as albumin, IgG, IgM, IgA,vitamin K-dependent-clotting factors and factor XIII (FXIII). Since theuse of plasma cryoprecipitate, as a source for the sealant, may resultin a lower activity of factor FXIII (Radosevich et al, 1997) some of thecompanies add exogenous factor XIII from various sources. Productsmanufactured from cryoprecipitate such as TissuCol (which has recentlyreceived a free marketing authorization in the US) and BeriPlast havebeen in the European market for more than a decade, however FXIII is notadded to any of these products in the U.S. Cohn fraction I, orequivalent products, are prepared by ethanol precipitation of plasma by8-10% ethanol at −30° C.-−50° C. at neutral pH. BioCol™ produced byPlasma Product services, Lille France is such product. The product ismanufactured by processing the whole plasma. Prior to a 10% cold ethanolprecipitation, the plasma is thawed at 37° C. for at least 6 hoursfollowed by recovery of the fibrinogen by centrifugation. The paste isthen reconstituted in Tris/lysine buffer and subjected to a second 10%cold ethanol precipitation for an additional 10-12 hours. This allowsfor highly purified fibrinogen, which is then treated by solventdetergent for virus inactivation. The final product is lyophilized forprolonged storage at 4° C. (Burnouf-Radosevich et al 1990).

Fibrinogen integrity and structure are of most importance for theperformance of fibrin sealant. The rates of polymerization, binding tothe fibrin receptor and susceptibility to fibrinolytic digestion have acritical influence on the performance in the clinical arena.

Fibrinogen, a tri-domainal (two D domains and a central E domain)disulfide bond molecule about 47 nm in length, is composed of twosymmetrical half-molecules, each consisting of a set of three differentpolypeptide chains terms Aa, Bb and γ. The halves are covalently joinedin the central amino-terminal E domain by five disulfide bridges.Reciprocal disulfide bridges are formed between the γ8 and γ9 position,thus orienting the γ chains in an anti-parallel direction (Mosesson,1997). Removal of the fibrinopepties A and B upon activation by thrombininduces a spontaneous polymerization of the fibrin monomer molecule vianoncovalentic binding.

Although fibrinogen has insufficient centers for such polymerization,nevertheless, in suitable conditions and without any enzymatic action,such as at high fibrinogen concentration, low ionic strength, or longincubation at 4° C., fibrinogen is polymerized (Rozenfeld and Vasileva1991). Fibrinogen is liable to undergo self-association according tosome authors to form structures that are either similar to fibrin (Cohenet al 1966; Gollwitzer et al 1983; Ugarova et al 1987) or toquasi-globular particles (Becker 1987).

Rozenfeld and Vasileva (1991) found that the molecules of fibrinogenundergo a spontaneous modification of their carboxyl terminals and bindend to end into flexible polymer chains. On attaining a critical length,the single-filament polymers twist into a coil and aggregate to formbranch molecules in which the segments are packed sufficiently denselyto resemble strong hydrated globular particles. The formation, under theinfluence of factor XIIIa, of ε/γ-glutamyl-lysine covalent bondsproduced only insignificant changes in the spatial organization of thefibrinogen aggregates. Covalent dimerization of the γ-chains restrictsthe structural flexibility of polymers, but linking of the a-chainsprovides progressive compaction of the structure with an increase in itsmolecular weight. Electrophoresis of reconstituted samples shows thatthe coil-shaped chains of fibrinogen oligomers prevent the completeenzymatic linking of the γ-chains. From the results of their work, theauthors suggest that the accelerated assembly of multimolecularaggregates, seen in the presence of Factor XIIIa, may be explained bythe stabilization of intermediate complexes of fibrinogen, which makethe spontaneous transition from a stable native state to the activestate irreversibly. However a more recent paper by Mosesson claims thatfibrinogen molecules become covalently crosslinked by factor XIIIa inthe same sequence as in fibrin, i.e. first the a and then the γ chain(Mega et al 1988, and Mosesson et al 1995).

It is an object of the present invention to provide a fibrin sealanthaving improved properties.

According to the invention fibrinogen multimers having at least 6fibrinogen units are used as an ingredient of a fibrin sealant.Preferably, 6-15 fibrinogen units in the multimer are used. Still morepreferred are 6-9 fibrinogen units. In particular 8 or 9 units arepresent in the multimer.

A method for manufacturing the fibrin sealant having fibrinogenmultimers with at least 6 units comprises the steps of

-   -   resuspending cryoprecipitate    -   adding sucrose to yield a concentration of 60 to 70% (w/w),    -   adding glycine to yield a concentration of 0.1 to 0.3 M,    -   heating to 60° C. for 15-20 hours,    -   removing the glycine and the sucrose by dialysis,    -   adding a protease inhibitor.

Preferably, the dialysis is conducted against a buffer comprising NaCl,glycine and CaCl₂.

Typically, the protease inhibitor is tranexamic acid in a concentrationof 8-12% (w/w) and/or arginine in a concentration of 1-3% (w/w).

Subject of the present invention is also a fibrin sealant comprisingmultimeric fibrinogen having at least 6 fibrinogen units.

In order to investigate the influence of the nature of the fibrinogencomponent in fibrin sealants, the immuno-reaction of the non-reducedagarose SDS electrophoresis method was modified first established byConnaghan et al (1985) and by Proietti et al (1990). The modification ofthis semi-quantitative method initially designed to monitor fibrincrosslinked polymers and its degradation products in plasma, was usedfor monitoring the number of fibrinogen monomers/units and their amountsin each oligomer in various fibrinogen preparations and in fibrinsealants.

Even though a detailed protocol for fibrinogen identification usingagarose horizontal gel electrophoresis has been published in 1990, themethod was unsatisfactory for the separation of high molecular wt.fibrinogen protein (Proietti et al 1990). The method developed by Reines(1990) for the detection of vWF multimers to be used for theimmunodetection of fibrinogen, was modified as follows: The HorizontalElectrophoresis was performed using the Reines et al (1990) detailedprotocol for vWF multimers, however, the detection of fibrinogenmultimers and the analysis method were of Proietti et al (1990), withsome modifications (Proietti et al 1990). Briefly, samples were preparedby diluting to a protein concentration of 0.1 mg/ml in a solution of 70mM Tris, 4 mM EDTA, 2.4% SDS, 9 M urea and 0.1 g/L bromophenol blueadded as a marker and incubated at 60° C. for 30 min. Agarose wasdissolved to a final concentration of 1% (stacking gel), 1.6% (cathodegel) and 2% (separating gel) by boiling in 70 mM Tris, 4 mM EDTA and 4%SDS (stacking gel buffer) or 100 mM Tris, 150 mM glycine and 0.1% SDS(cathode gel buffer and cathode buffer) or 200 mM Tris, 100 mM glycineand 0.4% SDS (separating gel buffer). Gels (cathode, stacking andseparating gels) were cast at >50° C. and allowed to polymerize for atleast 2 hours for each gel. Samples were loaded at 0.6-0.9 μl per well,then electrophoresis was performed toward the anode at 600-650volts/running hours at a constant temperature of 16° C. on a flat bedelectrophoresis apparatus (Pharmacia Fine Chemicals, Sweden). Therunning buffer was connected to the gel via 4-8 layers of filter paperimmersed in electrode buffer (the anode buffer had half the strength ofthe cathode buffer). Electrophoresis proceeded until the tracking dyehad migrated a distance of 6-8 cm. After electrophoresis, the gel wasremoved, the stacking and cathode gels taken away and the separating gelwashed in transfer buffer (25 mM Tris-HCl, 192 mM glycine pH 8.3 buffer,diluted 1:2 in 20% methanol) for approximately 1 minute.

Immunoblotting was performed onto a Nitrocellulose membrane (OptiranBA-S83 S&S Germany), cut to size, using the capillary blotting procedure(Reines et al 1990). After protein transfer, the nitrocellulose membranewas carefully removed from the capillary blotting unit and the residualprotein binding capacity blocked by immersion in blocking buffer (0.5%I-Block Tropix, USA, 0.05% Sodium Azide in PBS) for 1 hour.Immunodetection of fibrinogen multimers was done using a goat polyclonalanti-human fibrinogen antibody (Sigma, USA), and a rabbit polyclonalanti-goat immunoglobulin alkaline phosphatase conjugate (Sigma, Mo USA)was used for immunostaining.

SDS-Polyacrylamide electrophoresis was done in a verticalelectrophoretic unit (Hoefer Scientific Instruments, San Francisco,Calif. USA) using a reducing system containing 250 mM Tris, 40%glycerol, 4% SDS and 4% β-Mercaptoethanol (loading buffer). Theseparating gel was prepared as a gradient from 6 to 12% acrylamideconcentration (from a 30% acrylamide and 0.8% bisacrylamide stocksolution) containing 10% ammonium persulfate and 0.02% TEMED. Thesamples were mixed with 4× loading buffer to ¼ of the final volume andheated in a dry block at 95° C. for 5 minutes. The gel was run at 200Vand the electrophoresis was stopped when the Bromophenol Blue reachedthe bottom of the gel. Immunodetection was performed using a semi-dryimmuno-blotter (Hoefer Scientific Instruments, San Francisco, Calif.USA) onto a nitrocellulose membrane using the same antibodies andprotocol as for the horizontal electrophoresis.

FIG. 1 shows the immunodetection of fibrinogen multimers of three plasmafractionations in-process sources of fibrinogen. One source derived fromPaste I was supplemented with Factor FXIIIa. (From right to left) Lanes1-6: cryoprecipitate; Lanes 7-12: purified fibrinogen; Lanes 13-18:purified Fibrinogen+FXIIIa; Lanes 19-21: solvent detergent paste I;Samples were taken during the process of the invention; Lanes 1, 7, 13and 19: samples taken at the beginning of the process. Lanes 2, 8, 14and 20: samples taken after the addition of sucrose and glycine. Lanes3, 4, 9, 10, 15, 16 and 21: taken after incubation at 60° C. for 17hours. Lanes 5, 11, and 17: taken after dialysis and ultrafiltration.Lanes 6, 12, 18, and 22: taken after the formulation with 10% Tranexamicacid and 2% Argenine. All the samples were loaded at 0.7-0.9 μg perlane.

FIG. 2 shows the immunodetection of fibrinogen multimers electrophoresedon agarose SDS gel. Fibrinogen samples are of eight batches of theproduct of the present invention—lane 4: #C31102; lane 5: C30092; lane6: C29081; lane 7: C27071; lane 8: C25061; lane 9: C22052; lane 10:14032; lane 11: C12022. Four additional commercial sources of fibrinogenwere added—lane 1: Tisseel™; lane 2: BeriPlast; lane 3: BioCol and lane12: fibrinogen from Enzyme Research Inc. All the samples were loaded at0.65 μg per lane.

FIG. 3 shows the immunodetection of fibrinogen multimers of threecommercial preparations of fibrin sealants: From left to right. Lane 12:Tisseel™; lane 11: BioCol; lane 10: Fibrinogen from Enzyme researchInc.; lane 9: product prepared according to the method of the invention.Other lanes are fibrinogen production in process samples taken after thefollowing steps: lane 1: cryoprecipitate resuspension sample-BC01; lane2: aluminum alhydrogel absorption sample-BC02; lane 3: SDtreatment-BC04; lane 4: SD removal-BC06; lane 5: afterpasteurization-BC09; lane 6: addition of stabilizers-BC10; lane 7: finalconcentration-BC11, lane 8: sterile bulk-BC12. All the samples wereloaded at 0.65 μg per lane.

FIG. 4 shows a western blot-immunodetection of various formulations ofurea non-reduced fibrinogen multimers of product prepared according tothe method of the invention, run by Agarose-SDS gel electrophoresis.Lane 1: no Arginine and no Tranexamic acid; lane 2: 2% Arginine; lane 3:2% Arginine and 5% Tranexamic acid; lane 4: 2% Arginine and 10%Tranexamic acid; lane 5: 10% Tranexamic acid, lane 6: 1% Arginine and10% Tranexamic acid; lane 7: 4% Arginine and 10% Tranexamic acid, lane8: Fibrinogen from Enzyme Research Inc.; lane 9: Tisseel™; lane 10:BioCol. All samples were loaded at 0.65 μg protein per lane.

FIG. 5 shows western blot-immunodetection of various formulations ofreduced fibrinogen multimers of product prepared according to the methodof the invention, migrated on PAGE-SDS gel electrophoresis. Lane 1: NoArginine and no Tranexamic acid; lane 2: 2% Arginine; lane 3: 2%Arginine and 5% Tranexamic acid; lane 4: 2% Arginine and 10% Tranexamicacid; lane 5: 10% Tranexamic acid; lane 6: 1% Arginine and 10%Tranexamic acid; lane 7: 4% Arginine and 10% Tranexamic acid, lane 8:Fibrinogen from Enzyme Research Inc.; lane 9: Tisseel™. All samples wereloaded at 10 μg protein per lane.

The initial fibrinogen multimeric state in various fibrinogen sourcesand the effect of a production process on the polymerization wasidentified. Three fibrinogen sources were used: 1) Re-suspension ofcryoprecipitate (Sample A, Omrix Biopharmaceuticals LTD (Israel); 2)purified fibrinogen, derived from Cohn Fraction I (Sample B, EnzymeResearch, USA) or purified fibrinogen supplemented with FXIIIa (SampleC, Enzyme Research USA) and 3) solvent detergent treated fraction I(Sample D, from OctaPharma (Austria). They were treated with the processof the present invention. Pasteurization, dialysis and formulation withTranexamic acid and Arginine and at each stage of the procedure, sampleshave been taken to test the state of polymerization (see Table 1).

Samples labeled A1, B1, C1 and D1 are re-suspended fibrinogen samplesthat have been directly taken for testing (lanes 1, 7, 13 and 19 in FIG.1).

Samples labeled A2, B2, C2 and D2 are re-suspended fibrinogen samplesmixed with 65% (w/w) sucrose and 0.2 M glycine, incubated at 37° C. forless than 4 hours, heated to 60° C. to allow for complete sucrosedissolution (lanes 2, 8, 14 and 20 in FIG. 1).

Samples labeled A3, B3, C3 and D3 are re-suspended fibrinogen samplesmixed with sucrose and glycine, incubated at 37° C. for less than 4hours and incubated at 60° C. for 17 hours (lanes 3, 9, 15 and 21 inFIG. 1).

Samples labeled A4, B4 and C4 are re-suspended fibrinogen samples mixedwith sucrose and glycine, incubated at 37° C. for less than 4 hours.Then, incubation at 60° C. for 17 hours was performed and dialyzedagainst 10 mM citrate buffer 120 mM NaCl, 120 mM glycine and 1 mM CaCl₂(lanes 4, 10 and 16 in FIG. 1).

Samples labeled A5, B5, C5 and D5 are re-suspended fibrinogen samplesmixed with sucrose and glycine, incubated at 37° C. for less than 4hours, incubated at 60° C. for 17 hours, and dialyzed against 10 mMcitrate buffer 120 mM NaCl, 120 mM glycine and 1 mM CaCl₂. The resultingsolution was then formulated with 10% Tranexamic acid and 2% Arginine(lanes 5, 11 and 17 in FIG. 1). For additional clarifications see Table1.

TABLE 1 Experimental design of fibrinogen treatments, sources, additionof Factor XIIIa, incubation at high temperature, formulation during thepasteurization with sucrose and glycine and addition of final productstabilizers, Tranexamic acid and Arginine. The effect of all theseparameters on the fibrinogen multimeric state was tested. Procedure -Incubation Lane No. of end point of Incubation time at the # sample Nameof sample treatment temperature last point  1 A1 Cryoprecipitate — —   0 2 A2 Cryoprecipitate Sucrose, glycine 37° C.   4 h  3 A3Cryoprecipitate pasteurization 60° C.  17 h  4 A3′  5 A4 Cryoprecipitatedialysis and RT   3 h + 2.5 h ultrafiltration  6 A5 Cryoprecipitate add10% TEA, 2% RT   1 h Arginine  7 B1 Purified RT   0 Fibrinogen  8 B2Purified Sucrose, glycine 37° C.   2 h Fibrinogen  9 B3 Purifiedpasteurization 60° C.  17 h Fibrinogen 10 B3′ 11 B4 Purified dialysisand RT   3 h + 2.5 h Fibrinogen ultrafiltration 12 B5 Purified add 10%TEA, 2% RT   1 h Fibrinogen Arginine 13 C1 Purified RT   0 Fibrinogen +FXIII 14 C2 Purified Sucrose, glycine 37° C.   2 h Fibrinogen + FXIII 15C3 Purified pasteurization 60° C.  17 h Fibrinogen + FXIII 16 C3′ 17 C4Purified dialysis and RT   3 h + 2.5 h Fibrinogen + FXIIIultrafiltration 18 C5 Purified add 10% TEA, 2% RT   1 h Fibrinogen FXIIIArginine 19 D1 Fraction I S/D RT   0 20 D2 Fraction I S/D Sucrose,glycine 37° C. 1.5 h 21 D3 Fraction I S/D pasteurization 60° C.  17 h

FIG. 1 shows that the cryo-precipitate fibrinogen source has morefibrinogen multimers than the other fibrinogen sources, as it can beseen in bands 1, 7, 13 and 19. On the other hand, the 60° C. incubationincreased the number and the amounts of fibrinogen units per multimerand the percentage of multimeric fibrinogen. However, this effect ismost evident in purified fibrinogen and in Fraction I (lanes 9 and 15).The effect of FXIIIa addition is marginal, probably since all fibrinogensources from plasma contain traces of FXIII.

The effect of fibrinogen source is further demonstrated in FIG. 2. FIG.2 shows multimers of fibrinogen of fibrin sealant produced using theprocess of the invention, from cryoprecipitate after double virusinactivation (solvent detergent and pasteurization) and stabilizeraddition from eight batches. FIG. 2 also presents the Multimeric Stateof four fibrinogen concentrates from commercial sources (Purifiedfibrinogen Enzyme Research, TissuCol, Biocol and Beriplast). As can benoted from the figure, purified fibrinogen (Enzyme research Inc.)derived from Fraction I is primarily a monomer. However that preparationcontains a small amount of fibrinogen dimeric structure. On the otherhand, commercial fibrinogen products, which compose part of fibrinsealants, contain fibrinogen multimers up to pentamers while the fibrinsealant of the present invention has high multimeric fibrinogenstructures reaching up to octamers or nanomers. Furthermore, the batchto batch reproducibility, relative to the high multimeric state offibrinogen, is evident.

The first part of the study revealed that pasteurization has a majoreffect on the polymerization of fibrinogen from Fraction I. However, theeffect on cryoprecipitate was not fully demonstrated. In the second partof the study, the changes in the Multimeric State of fibrinogen duringthe production process and the batch to batch variability were examined.Samples were withdrawn from the process and the material was subjectedto examination by immunodetection after agarose gel electrophoresis.FIG. 3 shows fibrinogen immunodetection of samples obtained followingeach production step: cryo-resuspension—lane 1; alhydrogel absorptionlane 2; SD treatment—lane 3, SD removal—lane 4, post-pasteurization—lane5, stabilizer addition—lane 6, final concentration—lane 7, sterilebulk—lane 8 and final container—lane 9, purified fibrinogen—lane 10 andthe two commercial fibrinogen concentrates (Tissel and Biocol)—lanes 11and 12 respectively. The figure clearly shows that even though thecryo-resuspension contains six-mers (lane 1), the addition ofstabilizers (lane 6) promoted fibrinogen polymer formation since lane 6,sample after stabilizers addition, contains at least one more multimericunit than the previous sample, after pasteurization (line 5). Thisclearly indicates that the addition of Arginine and Tranexamic acid hadan effect on the fibrinogen multimeric state.

In order to examine the effect of each of the stabilizer, a sample waswithdrawn from the process prior to stabilizer addition. At this point,several combinations of Arginine and Tranexamic acid were formulatedcontaining between 0-4% and 0-10% (w/w) respectively. The differentformulations were frozen, stored for seven days and thawed at 37° C.After thawing, the samples, together with purified fibrinogen andcommercial fibrin concentrates, were run on a horizontal SDS-Agarose nonreduced and vertical reduced β-mercaptoethanol SDS-PAGE)electrophoresis. The results are shown in FIGS. 4 (horizontalSDS-Agarose non-reduced) and 5 (SDS-PAGE reduced β-mercaptoethanolSDS-PAGE). The horizontal agarose gel electrophoresis followed byspecific immunodetection of fibrinogen shows that addition of Argininepromotes multimer formation but the main contribution to its effect isdone by the addition of Tranexamic acid. This finding is supported bysamples that contains 0% Arginine and 10% TEA which produced more andlarger fibrinogen multimers than 2% Arginine and 0% TEA. The samephenomenon can be seen on the reduced β-mercaptoethanol SDS-PAGEconducted on the same samples (FIG. 5). A new band with a molecularweight above 250 kD appears in all formulations but not in theformulation with no stabilizers and not in the commercial concentratedfibrinogen tested (fibrinogen form Enzyme Research Inc and Tisseel™).Furthermore, in the samples formulated with 10% Tranexamic acid, thishigh molecular weight band is much more intense than other formulations,indicating an increase in the ratio of a high number of multimericunits. Another observation that can be made from the figure is theabsence of almost all the γ—γ and a-γ fibrinogen dimers from samplesproduced according to the method of the invention that are quiteabundant in other fibrinogen concentrates (Siebenlist and Mossesson,1996). This suggests that the increase of fibrinogen polymers is at theexpense of fibrinogen dimers. Moreover, the addition of Tranexamic acidand Arginine might stabilize the fibrinogen polymers or even drive thereaction towards a higher multimeric state.

It was established that native fibrinogen, left undisturbed, formsreversible temperature-dependent aggregates (Becker and Waugh, 1980;Becker, 1987). Based on light scattering and viscosity examinationsRozemfeld and Vasileva, (1991) suggested that fibrinogen undergoesspontaneous self-association through its D domain to form flexible endto end polymer chains. They found that self-association accrued morerapidly when factor XIIIa was present, but the spatial arrangement ofthe fibrinogen molecules remained the same. Masesson and Siebenlist 1995supply direct ultra-structural evidence for such associations(Siebenlist and Mossesson, 1996). However, the extent of these polymershas never been investigated in highly concentrated fibrinogen of thecommercial available fibrin sealants. Furthermore in this manuscript,for the first time, evidence is been given for a fractionationproduction process which might lead to such high multimeric appearanceof fibrinogen.

As noted in FIGS. 1 and 3, Cryoprecipitate production resulted in a highmultimeric structure. However, during the production process, some ofthe highest molecular multimers (seven to nine fibrinogen units) arelost, probably due to filtration and precipitation. Later steps in theprocess of the invention, such as pasteurization and stabilization withnegatively charged amino acid, recovered the high multimeric state andstabilized it even during a prolonged storage (see FIG. 2). Othercommercial product such as BeriPlast™ and Tisseel™ also derived fromcryoprecipitate, however, the final product contains lower multimericfibrinogen. Both contain 4-5 fibrinogen units whereas the productprepared according to the process of the invention contains about 8-9fibrinogen units. A different manufacturing process and differentformulation may explain this difference. Both are formulated withAprotinin and a low concentration of amino acid whereas the fibrinsealant prepared according to the process of the invention is formulatedwith a high concentration of Tranexamic acid and Arginine. This highsalt concentration probably facilitated the formation of the highmolecular wt. multimers. BioCol on the other hand is produced from amodification of Cohn fraction I. This starting material has a lowmultimeric state (see FIG. 1), probably because of the presence of ahigh concentration of ethanol which suppresses the enzymatic activity ofFXIII during the precipitation of fibrinogen.

The clinical relevance of the multimeric state has to be assessedcarefully by conducting well designed animal experiments. Since suchexperiments involve strong commercial interest, these experiments haveto be performed in a well-controlled manner. The animal model consistsof spraying of adhesive/fibrin sealant to control bleeding caused bypartial liver resection in heparinized rabbit. Results show that thebleeding time and the amount of glue used were significantly shorter andlower for the fibrin sealant prepared according to the process of thepresent invention than in other fibrinogen concentrate tested(BeriPlast™ and Tisseel™). These outcomes may indicate that it hasbetter adhesive properties to tissue than other glues. There are alsoreports indicating that it seems to work also at low temperature inindications such as liver transplantation. One explanation of the highadherence to tissue may be related to the high multimeric state of it ascompared to the other fibrin sealants tested. These large molecules,some of them of more than 2×10⁶ D molecular wt, are structurally verysimilar to the fibrin network. These protein macromolecules have betteradhesive forces than their monomeric units, very similar to thephenomenon known in vWF Multimers.

REFERENCES

-   1. Becker, C. M. (1987) Bovine fibrinogen aggregates: Electron    microscopic observations of quasi-globular structures, Thromb. Res.    48, 101-110.-   2. Becker, C. M., and Waugh, D. F. (1980) Bovine fibrinogens:    Reversible polymer formation, Arch. Biochem. Biophys. 204, 101-108.-   3. Burnouf-Radosevich M, Burnouf T, Huart J J: Biochemical and    physical properties of solvent-detergent-treated fibrin sealant. Vox    Sang 1990; 58:77-84.-   4. Cohen G, Stayer H, Kucera J Hall C, 1966 Polymorphism in    fibrinogen aggregates J Mol Biol 22: 385-388.-   5. Connaghan D G, Francis C W, Lane D A, Marder V J. Specific    identification of fibrin polymers, fibrinogen degradation products,    and crosslinked fibrin degradation products in plasma and serum with    a new sensitive technique. Blood 1985; 65:589-97-   6. Gladner J A, Nossal R. Effects of crosslinking on the rigidity    and proteolytic susceptibility of human fibrin clots Thromb Res    1983, 30:273-88-   7. Gollwitzer R, Bode W, Karges H E. On the aggregation of    fibrinogen molecules, Thromb Res Suppl 1983; 5:41-53-   8. Matras H: Fibrin seal: The state of the art. J. Oral1Maxillofac    Surg 1985; 43:605-611.-   9. Mega, J. I., Shainoff, J. R., and Dreshar, D. A. (1988)    Dissociation of α-fibrin at elevated temperatures, in Mosesson, M.    W., Amrani, D. L., Siebenlist, K. R., and DiOrio, J. P. (Eds.),    Fibrinogen 3. Biochemistry, Biological Functions, Gene Regulation    and Expression, pp. 83-86, Elsevier/North-Holland, Amsterdam.-   10. Mosesson M W. Fibrinogen and fibrin polymerization: appraisal of    the binding events that accompany fibrin generation and fibrin clot    assembly. Blood Coagul Fibrinolysis 1997 July; 8:257-67.-   11. Mosesson, M. W., Siebenlist, K. R., DiOrio, J. P., Matsuda, M.,    Hainfeld, J. F., and Wall, J. S. (1995). The role of fibrinogen D    domain intermolecular association sites in the polymerization of    fibrin and fibrinogen Tokyo II (γ 275 arg→cys), J. Clin. Invest. 96,    1053-1058.-   12. Proietti A B, McGuire M, Bell W. Specific identification of    fibrin(ogen) degradation products in plasma and serum using blotting    and peroxidase labeled antiserum. Am J. Hematol 1990; 34:270-274.-   13. Radosevich M, Goubran H I, Burnouf T. Fibrin sealant: scientific    rationale, production methods, properties, and current clinical use.    Vox Sang 1997; 72:133-43-   14. Raines G, Aumann H, Sykes S, Street A. Multimeric analysis of    von Willebrand factor by molecular sieving electrophoresis in sodium    dodecyl sulphate agarose gel. Thromb Res 1990; 60:201-12-   15. Rosenfel'd, M. A., and Vasil'eva, M. V. (1991) Mechanisms of    aggregation of fibrinogen molecules: The influence of    fibrin-stabilizing factor, Biomed. Sci. 2, 155-161.-   16. Siebenlist K R Mosesson M W. Evidence for intramolecular    cross-linked Aa*γ chain heterodimers in plasma fibrinogen.    Biochemistry 1996: 35:5817-5821.-   17. Sierra D H: Fibrin sealant adhesive systems: A review of their    chemistry, material properties and clinical applications. J.    Biomater Appl 1993; 7:309-352.-   18. Spotnitz W D, Mintz P D, Avery N, Bithell T C, Kaul S, Nolan S    P: Fibrin sealant from stored human plasma: An inexpensive and    efficient method for local blood bank preparation. Am Surg 1987;    53:460-464.-   19. Spotnitz W D: Fibrin sealant in the United States: Clinical use    at the University of Virginia. Thromb Haemost 1995; 74:482-485.-   20. Ugarova T P, Mikhalovskaia L I, Gornitskaia O V. Aggregation of    fibrinogen molecules in a solution. Ukr Biokhim Zh 1987; 59: 9-17.    [Article in Russian]-   21. Veklich, Y. Ang E K Loarnd, L, and Weisel J W. (1998). The    complementary aggregation sites of fibrinogen investigated through    examination of polymers of fibrinogen with fragment E. Proc. Natl.    Acad. Sci. 1998; 95:1438-1442.

1. A method for the production of a fibrin sealant comprising fibrinogenmultimers having at least 6 fibrinogen units comprising the steps ofobtaining a mixture by resuspending cryoprecipitate adding sucrosepowder to yield a sucrose/mixture concentration of 60 to 70% (w/w), andadding glycine powder to yield a glycine/mixture concentration of 0.1 to0.3 M, heating to 60° C. for 15-20 hours, removing the glycine and thesucrose by dialysis, and adding a protease inhibitor.
 2. The method ofclaim 1, wherein the dialysis is conducted against a buffer comprisingNaCl, glycine, and CaCl₂.
 3. The method of claim 1, wherein the proteaseinhibitor is tranexamic acid in a concentration of 8-12% (w/w) and/orarginine in a concentration of 1-3% (w/w).
 4. A fibrin sealantcomprising multimeric fibrinogen having at least 6 fibrinogen units. 5.The fibrin sealant of claim 4, wherein 6-15 fibrinogen units are presentin the multimeric fibrinogen.
 6. The fibrin sealant of claim 4, wherein6-9 fibrinogen units are present in the multimeric fibrinogen.
 7. Thefibrin sealant of claim 4, wherein multimeric fibrinogen having 6-9fibrinogen units is present in an amount of at least 5% (w/w) of totalfibrinogen content.